Fermentation and purification of mycolactones

ABSTRACT

Fermentation based methods for producing mycolactones allows one to produce large quantities of mycolactones, which can be purified by extraction and chromatography to yield pure preparations of mycolactones A, B, C, and D.

[0001] This application asserts priority to U.S. Provisional ApplicationNo. 60/222,649 filed Aug. 3, 2000 by inventors Peter Licari, RobertArslanian, Lawrence Cadapan and John Carney entitled FERMENTATION ANDPURIFICATION OF MYCOLACTONES which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention provides methods for preparing mycolactonesby fermentation and novel mycolactone compounds useful in the treatmentof disease conditions. The invention relates to the fields of chemistry,molecular biology, animal and human health sciences, and medicine.

BACKGROUND OF THE INVENTION

[0003]Mycobacterium ulcerans causes a severe skin disease, Buruli ulcer,which is characterized by extensive necrosis in the absence of an acuteinflammatory response. The causative agent of the disease was firstidentified as a diffusible toxic agent (see Read et al., June 1974,Cytotoxic activity of Mycobacterium ulcerans, Infection & Immun. 9(6):1114-1122, incorporated herein by reference). Further analysisidentified that the agent is a polyketide (see George et al., Feb. 5,1999, Mycolactone: A polyketide toxin from Mycobacterium ulceransrequired for virulence, Science 283: 854-857, incorporated herein byreference). Recently, the complete structural determination of twostructurally related agents, called mycolactones A and B, has beenreported (see Gunawardana et al., 1999, Characterization of novelmacrolide toxins, mycolactones A and B, from a human pathogen,Mycobacterium ulcerans, JACS 121: 6092-6093, incorporated herein byreference).

[0004] The cytotoxic nature of the mycolactones, as well as theirability to suppress the immune response, indicates that the compoundshave therapeutic potential as anti-cancer agents and immunosuppressants(see the references cited supra and Pimsler et al., Mar. 1988,Immunosuppressive properties of the soluble toxin from Mycobacteriumulcerans, J. Infect. Dis. 157(3): 577-580, incorporated herein byreference). However, despite extensive efforts to develop fermentationconditions suitable for large-scale growth of Mycobacterium ulcerans andrelated organisms such as M. bovis (see e.g. Mve-Obiang et al., 1999,Growth and cytotoxic activity by Mycobacterium ulcerans in protein-freemedia, FEMS Microbiol. Lett. 181: 153-157; Nyabenda et al., 1988, Theproduction of mycobacterial antigens by homogeneous culture in afermentor, J. Biol. Standard. 16: 259-267; Palomino & Portaels, Feb.1998, Effects of decontamination methods and culture conditions onviability of Mycobacterium ulcerans in the BACTEC system, J. Clin.Microbiol. 36(2): 402-408; and Palomino et al., Nov. 1998, Effect ofoxygen on growth of Mycobacterium ulcerans in the BACTEC system, J.Clin. Microbiol 36(11): 3420-3422, each of which is incorporated hereinby reference), cultivation of the organism remains difficult, limitingthe availability of the compounds for testing and clinical trials.

[0005] There remains a need for fermentation processes by which largescale cultures of Mycobacterium ulcerans can be obtained and from whichuseful quantities of mycolactones could be prepared. The presentinvention meets that need and provides as well, as one importantbenefit, novel naturally produced mycolactones in purified form thatwere heretofore undiscovered due to the lack of suitable fermentationprocesses.

SUMMARY OF THE INVENTION

[0006] In a first embodiment, the present invention provides methods forthe production of mycolactones by fermentation. The methods are readilyscalable and can be used to produce the mycolactones in amountssufficient for clinical trials and commercialization. The methods enablethe cultivation of Mycobacterium ulcerans in a dispersed suspensionculture (e.g., fermentation or spinner flasks) and the production of themycolactones at concentrations greater than that observed in T flasks.

[0007] In a second embodiment, the present invention provides improvedmedia formulations that allow scale-up of cultures for the production ofmycolactones and improve the production of mycolactone fromMycobacterium ulcerans.

[0008] In a third embodiment, the present invention also provides arobust and scalable method for the purification of mycolactones based onextraction and chromatography.

[0009] In a fourth embodiment the present invention provides novelmycolactone compounds isolatable from Mycobacterium ulcerans in purifiedform.

[0010] These and other embodiments, modes, and aspects of the inventionare described in more detail in the following brief description of thefigure, detailed description of the invention, the examples, and claimsset forth below.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 shows an HPLC chromatogram (top) and diode array spectra(bottom) of the solid phase extraction product of mycolactones preparedand purified in accordance with the methods of the invention. The HPLCchromatogram shows three distinct peak regions labelled F2 (fraction 2),F3 (fraction 3), and F4 (fraction 4). There are similar peak profileswithin each region with respect to relative peak intensities, retentiontimes, and diode array spectra. Fraction 3 contains mycolactones A andB. Fraction 2 contains a mixture of dehydrated mycolactones, termedmycolactone D. Fraction 4 contains a group of isomers with one lessoxygen than mycolactones A and B, termed mycolactone C.

[0012]FIG. 2 depicts an acid fast staining of a spinner flask culture.

[0013]FIG. 3a-b are comparisons of strains adapted and not adapted tosuspension culture. Spinner flasks (100 mL) were inoculated with aculture from a spinner flask () or stationary T-flask (▪). (A) Glucoseconcentration (g/L) and (B) mycolactone titers (mg/L) are plotted as afunction of time post inoculation.

[0014]FIGS. 4a-b are observations of new mycolactone congeners. Spinnerflask cultures in (FIG. 4a) M7H9/OADC or (FIG. 4b) M7H9/OADC containing2% egg yolk were analyzed 20 days after inoculation by LC-MS. Shown arethe elution profiles at 360 nm. The indicated regions were purified andconcentrated for mass spectral analysis and the predominant mass isgiven.

[0015]FIG. 5 depicts mycolactone production in stirred-tank fermenters.Shown are data for a 10 L working volume of M7H9/OADC (A) and a 150 Lworking volume of M7H9/OADC plus 2% egg yolk (B). Glucose concentration(g/L) (▪) and mycolactone titer (mg/L) () are shown as a function ofdays post-inoculation for both fermentations. Dry cell weight (g/L) (O)is shown only for the 10 L fermentation; egg yolk interfered with thismeasurement in the 150 L fermentation.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In a first embodiment, the present invention provides methods forthe production of mycolactones by fermentation. The methods are readilyscalable and can be used to produce the mycolactones in amountssufficient for clinical trials and commercialization. The methods enablethe cultivation of Mycobacterium ulcerans in a dispersed suspensionculture (e.g., fermentation or spinner flasks) and the production of themycolactones at concentrations greater than that observed in T flasks.

[0017] Prior to the present invention, Mycobacterium ulcerans was grownin stationary T-flasks to produce mycolactone; attempts to cultivate theorganism in suspension culture were unsuccessful. Using the methods ofthe present invention, one can grow the cells in a dispersed suspensionculture (e.g. fermentation or spinner flasks) and produce mycolactone inconcentrations greater than that observed in T flasks. This allows forscale-up of cultures for the production of mycolactones in commerciallyrelevant quantities.

[0018] In a second embodiment, the present invention provides improvedmedia formulations that allow scale-up of cultures for the production ofmycolactones and improve the production of mycolactone fromMycobacterium ulcerans. The improved media formulations increase theamount of mycolactones produced in the culture, again facilitating theproduction of mycolactones in commercially relevant quantities.

[0019] In a third embodiment, the present invention also provides arobust and scalable method for the purification of mycolactones based onextraction and chromatography. Prior to the present invention,mycolactone was purified by TLC. While one can obtain sufficient amountsof mycolactone using TLC-based purification to determine its structureand conduct limited in vitro and ex vivo testing, such methods are notpractical for the production of mycolactones in amounts required forpre-clinical in vivo testing and for clinical trials.

[0020] In a fourth embodiment the present invention provides novelmycolactone compounds isolatable from Mycobacterium ulcerans in purifiedform. The improved fermentation methods and media formulations as wellas the improved purification methods of the invention have enabled thedetection, isolation, and purification of novel mycolactone compoundsproduced in M. ulcerans that have previously not been detected. Two suchnovel compounds are described below. Both are produced at lower levelsthan mycolactones A and B, and one of the novel compounds, designatedmycolactone C, is produced at a significantly higher level than theother, designated mycolactone D.

[0021] Mycolactone C has a molecular mass 16 daltons lower thanmycolactone A and B, indicating the loss of an oxygen atom relative tothe known compounds. Initial characterization of mycolactone C indicatesthat the absent oxygen atom is from the fatty acid like portion ofmycolactone A and B. Initial characterization of mycolactone D indicatesthat these compounds are dehydration products of mycolactones A and B.

[0022] Thus the novel methods of the invention for the production andpurification of the mycolactones have enabled not only the production ofmycolactones A and B in quantities suitable for testing but also theidentification of previously unidentified mycolactones C and D.

[0023] The compounds of the invention can be readily formulated toprovide the pharmaceutical compositions of the invention. Thepharmaceutical compositions of the invention can be used in the form ofa pharmaceutical preparation, for example, in solid, semisolid, orliquid form. This preparation will contain one or more of the compoundsof the invention as an active ingredient in admixture with an organic orinorganic carrier or excipient suitable for external, enteral, orparenteral application. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, pessaries, solutions,emulsions, suspensions, and any other form suitable for use.

[0024] The carriers which can be used include water, glucose, lactose,gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate,talc, corn starch, keratin, colloidal silica, potato starch, urea, andother carriers suitable for use in manufacturing preparations, in solid,semi-solid, or liquified form. In addition, auxiliary stabilizing,thickening, and coloring agents and perfumes may be used. For example,the compounds of the invention may be utilized with hydroxypropylmethylcellulose essentially as described in U.S. Pat. No. 4,916,138,incorporated herein by reference, or with a surfactant essentially asdescribed in EPO patent publication No. 428,169, incorporated herein byreference.

[0025] Oral dosage forms may be prepared essentially as described byHondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22,incorporated herein by reference. Dosage forms for external applicationmay be prepared essentially as described in EP Pub. No. 423,714,incorporated herein by reference. The active compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the disease process or condition.

[0026] For the treatment of conditions and diseases caused by infection,immune system disorder (or to suppress immune function), or cancer, acompound of the invention may be administered orally, topically,parenterally, by inhalation spray, or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvant, and vehicles. The term parenteral, asused herein, includes subcutaneous injections, and intravenous,intrathecal, intramuscular, and intrasternal injection or infusiontechniques.

[0027] Dosage levels of the compounds of the present invention are ofthe order from about 0.01 mg to about 100 mg per kilogram of body weightper day, preferably from about 0.1 mg to about 50 mg per kilogram ofbody weight per day. The dosage levels are useful in the treatment ofthe above-indicated conditions (from about 0.7 mg to about 3.5 mg perpatient per day, assuming a 70 kg patient). In addition, the compoundsof the present invention may be administered on an intermittent basis,i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.

[0028] The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Forexample, a formulation intended for oral administration to humans maycontain from 0.5 mg to 5 μm of active agent compounded with anappropriate and convenient amount of carrier material, which may varyfrom about 5 percent to about 95 percent of the total composition.Dosage unit forms will generally contain from about 0.5 mg to about 500mg of active ingredient. For external administration, the compounds ofthe invention may be formulated within the range of, for example,0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, andmost preferably from about 0.005% to 0.8% by weight.

[0029] It will be understood, however, that the specific dose level forany particular patient will depend on a variety of factors. Thesefactors include the activity of the specific compound employed; the age,body weight, general health, sex, and diet of the subject; the time androute of administration and the rate of excretion of the drug; whether adrug combination is employed in the treatment; and the severity of theparticular disease or condition for which therapy is sought.

[0030] In another embodiment, the present invention provides a method oftreating cancer, which method comprises administering a therapeuticallyeffective amount of a novel epothilone compound of the invention.

[0031] The following examples are given for the purpose of illustratingthe present invention and shall not be construed as being a limitationon the scope of the invention or claims.

EXAMPLE 1 Production of Mycolactones by Fermentation

[0032] A. Production in Plates

[0033] A primary plate is prepared as follows. A 2 mL culture ofMycobacterium ulcerans (161 SWT, from NIH, but any mycolactone producingMycobacterium species can be employed in accordance with the methods ofthe invention) is spread evenly on a 150×15 mm M7H9 agar plate using asterile spreader. The plate is incubated at 30° C. for 4-8 weeks or whensignificant growth (yellow, waxy colonies) appears on the plate.Secondary plates can be made by streaking colonies from the primaryplate or from liquid culture.

[0034] B. Production in T-Flasks

[0035] A primary T-flask is prepared as follows. About 10 mL of M7H9liquid medium is poured onto the surface of a plate (see above)containing Mycobacterium ulcerans colonies. A sterile spreader is usedto loosen the colonies from the agar, and a sterile serological pipetteis then used to transfer the loose colonies onto a 600 mL T-triple flaskcontaining 300 ml of M7H9 medium. The T-flask is then incubated at 30°C. for 4-8 weeks or until significant growth (yellow, clumpy cells) isobserved. In a typical experiment, the amount of mycolactones recoveredfrom 1.8L of broth using M7H9 medium was 3 mg, and the approximate titerwas 3 mg/1.8 L or 1.7 mg/L.

[0036] Secondary or successive T-flasks can be made using culture fromthe primary T-flask or other T-flasks by the addition of 100 mL ofculture to 200 mL of M7H9 medium.

[0037] C. Production in Spinner Flasks

[0038] M7H9 medium is added to a spinner flask at a desired volume andinoculated with liquid culture from T-flasks at a 10% inoculum volume.The spinner flask is placed in a 30° C. warm room on a magnetic stirringplate and stirred at a speed that will result in adequate mixing to keepcellular clumps in suspension. The spinner flask is incubated for 4-8weeks or until mycolactone concentration detected in the broth reaches aplateau.

[0039] A variety of experimental parameters have been tested, including:80-100 mL total volume in a 100 mL spinner flask (Bellco) agitated at300-400 rpm; 175-210 mL total volume in a 250 mL spinner flask (Bellco)agitated at 200-300 rpm; 300-350 mL total volume in a 500 mL spinnerflask (Bellco) agitated at 150-250 rpm; 6.7 L total volume in a 15 Lspinner flask (Bellco) agitated at 100-150 rpm; and 10 L total volume ina 15 L spinner flask (Coming) agitated at 50-100 rpm. The amount ofmycolactones recovered from 6 L of broth from a 15L spinner flask usingM7H9 medium with 2% egg yolk enrichment was 28 mg, yielding anapproximate titer of 28 mg/6 L or 4.7 mg/L. Optical density (O.D.) wasused to monitor cell density and growth of the bacterial culture using aspectrophotometer at a wavelength of 600 nm.

[0040] D. Production in Fermentor

[0041] M7H9 medium is added to a fermentor at a desired volume andinoculated with liquid culture from T-flasks or spinner flasks at a 10%inoculum volume. The culture is maintained at the following parametersfor the duration of the run: pH=6.5+/−0.1, temperature=30+/−0.1° C.,dissolved oxygen=50% +/−20%, airflow=<1 LPM, agitation=500+/−200 rpm.The pH is controlled via addition of acid (2.5N H₂SO₄) and base (2.5NNaOH). The duration of the fermentor run is for 4-8 weeks or untilmycolactone concentration detected in the broth reaches a plateau. Theamount of mycolactones recovered from 8.5 L of broth from a 10 Lfermentor (B. Braun Biostat B) was 30 mg, yielding an approximate titerof 3.5 mg/L. Dry cell weight (DCW) was used to monitor the cell densityand growth of the bacterial culture.

[0042] The DCW procedure is as follows.

[0043] 1) Place 40 mL of well-mixed broth in a 50 mL conical falcon tube(tared).

[0044] 2) Spin down at 3300 g in a centrifuge for 10 minutes.

[0045] 3) Decant supernatant.

[0046] 4) Add 40 mL of water to pellet and resuspend by shaking orvortexing.

[0047] 5) Spin down again at 3300 g in a centrifuge for another 10minutes.

[0048] 6) Place tube in an 80° C. oven for >24 hours and re-weigh.

[0049] 7) Calculate DCW by: [(Dried Pellet+Tube)−(Tare of Tube)] /40 mL

[0050] In one fermentor experiment, the maximum DCW achieved was 1.4g/L.

EXAMPLE 2 Mycolactone LC/MS Analysis

[0051] For analysis of mycolactone by LC/MS, add 1 mL of methanol to 1mL of broth in a 1.5 mL eppendorf tube. Spin the sample using amicrocentrifuge set at 13,000 rpm for 5 minutes. Remove the MeOHextract, and filter it through a 0.45 gm syringe filter if necessary (ifextract is cloudy and contains particulates), and place the extract intoa sample vial for LC/MS analysis.

EXAMPLE 3 Harvest of Mycobacterium ulcerans Culture

[0052] To harvest the culture, measure the volume of culture broth andto it add an equal volume of MeOH. Let the solution mix for 20-30 min.Separate the solids from the extract by either pouring broth intocentrifuge bottles and centrifuging for 30 min. at 10,800 g or by usinga depth filter. The extract is then ready for purification.

EXAMPLE 4

[0053] Medium Preparation M7H9 Medium Component Concentration (g/L)Ammonium Sulfate 0.5 Monopotassium Phosphate 1.0 Disodium Phosphate 2.5Sodium Citrate 0.1 Magnesium Sulfate 0.05 Calcium Chloride 0.0005 ZincSulfate 0.001 Copper Sulfate 0.001 L-Glutamic Acid 0.5 Ferric AmmoniumCitrate 0.04 Pyridoxine 0.001 Biotin 0.0005 Glycerol 2 ml/L

[0054] To prepare the M7H9 medium, combine the M7H9 medium components in900 mL of water, sterilize by autoclaving at 121° C. for 30 min., coolto room temperature, and add 100 mL of OADC enrichment. M7H9 medium canbe obtained in a pre-formulated powder form (Difco) to which 4.7 g ofthe powder is added to 900 mL of water with the addition of 2 mL ofglycerol. The medium is sterilized as described, and 100 mL of OADCenrichment is added. Autoclave time increases as the volume of themedium increases (i.e., autoclave time for 10 L medium in a spinnerflask is 120 min.). OADC Enrichment Component Concentration (g/L) OleicAcid 0.5 Albumin Fraction V, Bovine 50.0 Dextrose 20.0 Catalase (Beef)0.04 Sodium Chloride 8.5

[0055] To prepare OADC enrichment, combine the above components in waterand bring the volume to 1 L. Sterilize the mixture with a sterile filterhousing with a 0.2 μm nylon membrane. OADC enrichment can also beobtained in pre-formulated liquid form (BBL).

[0056] For M7H9 agar (used in plates), 15 g/L of Bacto agar is added toM7H9 before autoclaving. The medium is then autoclaved as describedabove and allowed to cool to 50-55° C., at which time 100 mL of OADC areadded. The medium is poured onto 150×15 mm plates before it solidifies.

[0057] The present invention also provides new media formulations thatincrease growth rate and production of the mycolactones. One suchimproved formulation is prepared by doubling the concentration of M7H9components, including OADC enrichment. Another formulation involves theaddition of 50% egg yolk enrichment (Difco), which comes in liquid form,to M7H9 medium as a post-sterile addition at a concentration of 20 ml/Lor 2%.

EXAMPLE 5 Purification of Mycolactones

[0058] Mycolactones A and B were isolated as an isomeric mixture (28 mg)from 6 L of Mycobacterium ulcerans culture broth. Two new compounds,also as isomeric mixtures, were also isolated. HPLC (diode array) andLC/MS data indicate that the more abundant of the two new compounds,designated mycolactone C, is a desoxymycolactone, while the minorcomponent is a mycolactone dehydration product.

[0059] Whole broth (6 L) was mixed with an equal amount 100% methanol(Fisher bulk). The mixture was centrifuged (1500×g, 5 minutes) and thecentrate loaded onto a HP20 (Mitsubishi with SPE resin) capture column(4.8×25 cm). The column was eluted with 3 L of 100% methanol, which wasevaporated to dryness. The resulting solids were extracted twice with100 mL of 100% methanol. Following filtration the filtrates werecombined and evaporated, giving 1.16 g of a dark oil. The oil wasdissolved in 20 mL of 100% methanol to which 20 mL of deionized waterwere added. The resulting suspension was chromatographed on a 1×25 cmC18 (Bakerbond, 40 μm C18 resin) chromatography column previouslyequilibrated with 50% methanol in water. Column elution was carried outin a step gradient fashion starting with 80%, then 90%, and finally 100%methanol. Five fractions were collected. Fraction changes were made withthe aid of on-line UV monitoring at 360 nm. Fractions were evaporatedand re-dissolved in 10 mL of acetone, filtered and dried.

[0060] HPLC data of the solid phase extraction product showed threedistinct peak regions (see FIG. 1). Examination revealed similar peakprofiles within each region with respect to relative peak intensities,retention times, and diode array spectra. C18 column chromatography wasused to separate the three regions into three fractions. Fraction 3 (28mg) contained mycolactones A and B. Mass spectra data on fraction 2indicated a mixture of dehydrated mycolactones, while data for fraction4 indicated a group of isomers with one less oxygen than mycolactones Aand B.

[0061] The structure of mycolactone B is shown below.

[0062] In another example, mycolactones A and B were isolated as anisomeric mixture (32 mg) from 10.3 L of Mycobacterium ulcerans broth.HPLC (UV 360 nm) data showed a complex mixture of peaks corresponding tomycolactones A and B as well as other minor isomers. Compoundidentification was made on the basis of HPLC (diode array), LC/MS, andNMR data obtained on a mycolactone A and B mixture. Individualmycolactones were isolated as enriched isomeric fractions usingsemi-preparative HPLC. When samples were shielded from laboratory light,the individual mycolactones were stable. Samples exposed to laboratorylighting showed substantial isomerization after 52 hours.

[0063] Whole broth (1.8 L) was mixed with an equal amount 100% methanol(Fisher ACS grade). The mixture was allowed to settle for 30 minutes.The mixture was decanted and the resulting solution loaded onto a solidphase extraction column (4.8×25 cm) containing HP20 (Mitsubishi HP20 SPEresin) previously equilibrated with 50% methanol in water. Loading ofthe extract was carried out at 100 mL/minute. The column was eluted with3 L of 100% methanol in water. The first 250 mL were discarded while theremainder of the eluant was collected as a single fraction. The solidphase extraction product pool (2.5 L) was evaporated to dryness using aBuchi R153 (20 L, 40° C.) rotary evaporator (“rotovap”). This gave a redglass, which was extracted with 100 mL of 100% methanol in water,filtered through Whatman #4 filter paper, and evaporated to dryness(vacuum oven, 10 mbar, 40° C.). The dry material (280 mg) was dissolvedin 50 mL of 100% methanol to which 50 mL of water were added. Thesuspension was loaded onto a 1×25 cm C18 (Bakerbond C18 resin, 40 μg)chromatography column previously equilibrated with 50% methanol inwater. Loading and elution flow rate was 4 mL/minute. The column waseluted with 120 mL of 80% methanol in water followed by 120 mL of 90%methanol in water. A total of 24×10 mL fractions were collected withfractions 12-16 being combined and taken as the best pool. Evaporationgave 11 mg of a yellow oil. This material was triturated with 2 mL ofacetone, filtered through a 0.25 um filter, and evaporated to give 3 mgof a yellow oil.

[0064] In another example, whole broth (8.5 L) was mixed with an equalamount of 100% methanol. The mixture was centrifuged (30 minutes at10,800×g), decanted, and the resulting extract loaded onto a solid phaseextraction column (4.8×25 cm) containing HP20 previously equilibratedwith 50% methanol in water. Loading of the extract was carried out at100 mL/minute. The column was eluted as one fraction with 5 L of 100%methanol. The methanol eluant was evaporated to dryness using a 20 LBuchi rotovap, and the resulting solids were extracted with 100 mL of100% methanol, vacuum filtered through Whatman # 4 filter paper, andevaporated using a R124 Buchi rotovap (2 L, 40° C.). The oil wasdissolved in 50 mL of 100% methanol and diluted with 50 mL of water toform a suspension, which was loaded onto a pre-equilibrated 1×25 cm CC18 chromatography column. The column was eluted with 240 mL (12 columnvolumes) of 85% methanol. A total of 24×10 mL fractions were collectedand assayed by HPLC. Fractions 10-16 were combined and evaporated usinga R124 Buchi rotovap to give 42 mg of a yellow oil. The oil wastriturated with dichloromethane, filtered through glass wool and driedto give 36 mg of a yellow oily material. This material was trituratedwith 10 ml of acetone, filtered through a small plug of glass wool, andevaporated using a R124 Buchi rotovap. This gave 30 mg of a yellow oil.Mycolactone mixtures, each containing mycolactone A and B, were combinedto give a 33 mg of a yellow oil.

[0065] The individual components of the mycolactone mixture wereseparated using semi-preparative HPLC. The column used was an InertsilODS3 5 μm (1×25 cm) semi-preparative HPLC column. A gradient HPLC methodstarting with 65:35 acetonitrile in water and ending with 100%acetonitrile was used over a run time of 16 minutes. Four 100 μLinjections were used. 16 mg of material were used as starting material.For each run, three fractions were collected. Fraction changes werebased on on-line monitoring at 400 nm.

[0066] In another example, an enriched fraction (33 mg) containingmycolactone A and B isomers was isolated from a total of 10.3 L of wholebroth. HPLC diode array data of the enriched fraction showed a complexmixture of peaks all containing a primary UV maximum at 360 nm and asecond maximum at 260 nm. The strong absorbance at 360 nm was diagnosticfor the mycolactones and allowed for tracking of the various isomers.HPLC (diode array), along with LC/MS and NMR data confirmed the presenceof mycolactones A and B in this mixture. Semi-preparative HPLC wascarried out on 16 mg of the isomeric mixture and yielded 2 substantiallyenriched isomeric fractions. The enriched samples were shielded fromlaboratory light. Evaporation of pools B and D gave 2 and 4 mg of yellowoils.

[0067] To monitor stability of the individual enriched isomers, HPLCdata was collected on samples stored in the presence and absence oflaboratory light. Measurements were made on samples stored in methanolat 23° C. Initial HPLC sampling was carried out on individual isomersshielded to minimize exposure to laboratory light. These same sampleswere allowed to remain in the autosampler, shielded from light, for 72hours before additional HPLC data was collected. Following this, thesamples were stored on a lab bench exposed to laboratory light for 52hours, at which time the final HPLC data time point was collected. Underthese conditions, samples that were shielded from light remained stable,showing no change in their respective HPLC chromatograms. Howeversamples exposed to light for 52 hours showed substantial isomerization.

EXAMPLE 6 Production of Mycolactones

[0068] Like other pathogenic Mycobacteria, M. ulcerans grows slowly,with a doubling time of 1-2 days, and the cells form aggregates inliquid media. Cultivation of M. ulcerans for 4-6 weeks in stationaryT-flasks containing M7H9/OADC medium yields a final dry cell weight ofca. 1.4 g/L and mycolactone titers of 4-6 mg/L (George, K. M., et al.,Sci. (1999) 283:854-857), a cumbersome process for producing thecompound. Here we describe the adaptation of M. ulcerans to suspensionculture in spinner flasks and development of a scalable process forproducing mycolactones in larger quantities. A fed-batch fermentationprocess was implemented up to the 150 L scale and several newmycolactone congeners were identified.

[0069] MATERIALS AND METHODS

[0070] Strain and Media. Mycobacterium ulcerans, strain 1615 (TrudeauCollection Strain, Lake Saranac, N.Y.) was obtained from Dr. Pam Small.The culture was initially grown on M7H9/OADC agar plates at 30° C. for4-8 weeks (until yellow colonies were well formed). Cultures inM7H9/OADC medium were brought to 15% glycerol and samples were stored at−80° C. to provide a consistent source of inocula.

[0071] Difco M7H9 powder (4.7 g) was dissolved in 900 mL deionized watercontaining 2 mL/L glycerol and sterilized by autoclaving at 121° C. for30 min. After autoclaving, the medium was cooled to room temperaturebefore OADC enrichment (oleic acid, albumin, dextrose, and catalase,pre-formulated from Difco) was added to a concentration of 100 mL/L ofmedium. To prepare plates, 15 g/L agar was added to the M7H9 basalmedium before autoclaving and plates were poured immediately afteradding OADC enrichment to the medium at 50° C. In some experimentsliquid egg yolk enrichment (Difco) was added to a final concentration of20 mL/L (Palomino, J. C., et al., {J Clin. Microbiol. (1998)36:402-408).

[0072] T-Flask cultivation. To a plate containing M ulcerans coloniesM7H9/OADC medium (10 mL) was added and a sterile spreader was used todetach the cells. The suspended cells were transferred into a 600 mlT-flask (triple layer, Nunc) containing 300 mL of M7H9/OADC medium andthe flask was incubated at 30° C. for 4-8 weeks (until significantgrowth was observed). Cultures were expanded by adding 100 mL of cultureto 200 mL of fresh M7H9/OADC medium.

[0073] Growth in spinner flasks. A spinner flask containing the desiredvolume of M7H9/OADC medium was inoculated with a 10% volume of liquidculture and incubated at 30° C. for 4-8 weeks, or until mycolactoneproduction ceased. Conditions depended on the spinner flask used (Bellco100 ml spinner: 80-100 ml working volume, 300-400 rpm; Bellco 250 mlspinner: 175-210 ml working volume, 200-300 rpm; Bellco 500 ml spinner:300-350 ml working volume, 150-250 rpm; Bellco 15 L spinner: 6.7 Lworking volume 100-150 rpm; Coming 15 L spinner: 10 L working volume,50-100 rpm).

[0074] Growth in stirred-tank fermenters. M7H9/OADC medium, with orwithout 2% egg yolk, was used in fermentation studies. The followingparameters were maintained: pH 6.5∓0.1; 30° C.±0.1° C.; 50%±20%dissolved oxygen; airflow ≦1 LPM; 500±200 rpm agitation. The pH wascontrolled by adding 2.5N H₂SO₄ or 2.5N NaOH. Fermentation continued for4-8 weeks until mycolactone production had ceased. The glucoseconcentration in culture samples was measured using a YSI glucoseanalyzer.

[0075] Analysis of mycolactones. Whole culture broth samples wereprepared for analysis by adding an equal volume of methanol and removinginsoluble material by centrifuging at 12,000 g. Mycolactones wereresolved and quantitated using a Hewlett Packard 1090HPLC with UVdetection at 360 nm. Supernatant (250 μl) was injected and themycolactones captured on a 4.6×10 mm column (Inertsil, C18 ODS-3, 5 pm).After washing with 50% acetonitrile for 2 minutes the mycolactones wereeluted and resolved on a 4.6×150 mm column (Inertsil, C18 ODS-3, 5 μm)with a 24 minute gradient from 50% to 100% acetonitrile. In some cases,mycolactones were analyzed using a system comprised of a Beckman SystemGold HPLC, an Alltech ELSD detector, and a PE SCIEX API100LC MS-baseddetector equipped with an atmospheric pressure chemical ionizationsource. Fractions of interest were analyzed on a Applied BiosystemsMariner time-of-flight mass spectrometer with a turbo ion-spray source.

[0076] Other microbiological methods. Dry cell weight was determined bycentrifuging a 40 mL sample of culture for 10 minutes at 3300 g in apre-weighed 50 mL centrifuge tube, decanting the supernatant, washingthe cell pellet with 40 mL water, drying in an 80° C. oven, and weighingthe pellet. Acid fast staining was done with the Acid-Fast BactiStainkit from Polysciences according the manufacturer's instructions.

[0077] Results And Discussion

[0078] Adaptation of the Cells to Growth in Suspension

[0079] For a scalable process, it was necessary to adapt the cells togrowth in suspension. T-flask cultures were used to inoculate M7H9/OADCmedium in a variety of vessel configurations, including baffled flasks,flasks fitted with stainless steel springs, and spinner flasks designedfor growth of suspension-adapted animal cells. Because more dispersedgrowth of M ulcerans has been reported with addition of Tween 80(Mve-Obiang, A., et al., FEMS Microbiol. Lett. (1999) 181:153-157;Power, D. A., et al., Am. Rev. Resp. Dis. (1965) 92:83-93), this wasalso investigated. All the shake flask conditions tested, with orwithout Tween 80, gave severe aggregation of the cells, poor growth, andnegligible mycolactone production. Surprisingly, however, spinner flaskcultures gave dispersed growth. Acid-fast staining and microscopicexamination (FIG. 2) shows that the cultures were mostly individual rodswith a few clumps consisting of only a few cells. Such dispersed growthhas not been reported previously for any slow growing mycobacterialspecies.

[0080] To demonstrate that the cells had been adapted to dispersedgrowth, identical spinner flasks were inoculated with a 10% volume ofculture from a T-flask and from a spinner flask. By comparing the ratesof glucose consumption and mycolactone production in these cultures(FIG. 3), it is clear that significant adaptation occurred during growthin the spinner flask.

[0081] Cell density was difficult to measure and an accurate growth ratecould not be obtained from the spinner flask cultures. Also, degradationof mycolactones was often observed at later time points, indicating theimportance of monitoring production closely to permit harvest at themaximum titer. Although low oxygen tension has been reported to enhanceM. ulcerans growth (Palomino, J. C., et al., J. Clin. Microbiol. (1998)36:3420-3422), we did not observe any obvious enhancement in growth byreducing air flow.

[0082] Detection of Additional Mycolactones

[0083] Mycolactones A and B are double bond isomers with the same mass(Gunawardana, G., et al., J. Am. Chem. Soc. (1999) 121:6092-6093). Underour reverse-phase HPLC conditions, these isomers are resolved and elutein the 12.0-12.5 minute region (FIG. 4a). The order that A and B elutehas not been established and another peak was observed between A and Bwhich represents yet another mycolactone isomer by LC/MS analysis.Although the structure of this new isomer has not been determined, wepresume it represents isomerization at a different double bond. Additionof egg yolk to mycobacterial media has been reported to be beneficial(Palomino, J. C., et al., J. Clin. Microbiol (1998) 36:402-408) and wefound this addition generally increased mycolactone titers. Moreover,new compounds with the same UV spectrum as mycolactones A and B werereadily observed which eluted from the column at significantly differentretention times (FIG. 4b). Material that eluted in the regions around9.7 and 13.5 minutes was separately collected and analyzed by massspectrometry. The material eluting around 13.5 minutes gave a prominent[M+Na]+at m/z 749.5 and a weak pseudo-molecular ion at m/z 727.5,corresponding to a mycolactone congener with one less oxygen atom. Thematerial eluting around 9.7 minutes gave a prominent ion at m/z 747.5,which presuming it is also a sodium adduct, would correspond tomycolactones A and B with one less oxygen atom and two less hydrogenatoms. Although these new mycolactone congeners could be detected whenthe strain was cultivated in the absence of egg yolk, its additiongreatly facilitated the analysis. The masses of these new mycolactonecongeners are consistent with compounds that could arise by differentβ-keto processing on a putative type I PKS. The difference of an oxygenatom could, alternatively, be due to a difference in the extent of aputative hydroxylase reaction. The ratio of areas for each group ofpeaks eluting at the three regions are very similar (FIG. 4b),suggesting that the same equilibrium mixture of double bond isomers isreached for each congener. Equilibration between mycolactones A and Bwas found to occur only when the sample was exposed to ambient light(data not shown), which is consistent with double bond isomerization.

[0084] Scale-up to Stirred-tank Fermenters

[0085] Based on success with the spinner flasks, fermentations were alsocarried out in stirred tank fermenters. FIG. 5a shows a fermentation ina 10 L fermenter using a 400 mL inoculum (4%) from a spinner flaskculture. In this fermentation there was a long lag period before glucoseutilization and mycolactone production began. However, additional workin spinner flasks showed that larger inoculum volumes greatly reducedthis lag period. Mycolactone production ceased at approximately the sametime that glucose was depleted from the medium, suggesting that addingmore glucose could be beneficial.

[0086] Using data from the 10 L fermentation and several spinner flaskexperiments, M. ulcerans cultivation was scaled up to a 150 L fermenterusing M7H9/OADC supplemented with 1% egg yolk (FIG. 5b). A 10% inoculumwas used to reduce the lag phase. The glucose concentration decreased to<0.5 g/L on day 17, at which point additional glucose was added toprevent carbon limitation. Glucose continued to be consumed at a rapidrate until about day 26. The additional glucose may have caused theincrease in mycolactone production. Since more than 2 g/L of glucoseremained in the culture when growth had ceased, another essentialnutrient may become limiting. Although we have developed a scalableprocess, mycolactone titers were essentially the same as observed inT-flasks. However, this process is amenable to further development thatshould lead to increased titers.

[0087] Recovery of Mycolactones from the Culture

[0088] The distribution of mycolactones between the solid and solublefractions of culture broth was analyzed in samples from M7H9/OADC withor without added egg yolk. The distribution between pellet andsupernatant fractions was approximately equal with M7H9/OADC alone, butaddition of egg yolk shifted the distribution such that over 95% of themycolactones were in the pellet fraction. These data suggest thatmycolactones are secreted from the cells, but remain associated withsuspended lipids. Thus, in addition to enhancing titer, supplementationwith egg yolk can simplify downstream processing.

[0089] The invention having now been described by way of writtendescription and examples, those of skill in the art will recognize thatthe invention can be practiced in a variety of embodiments and that theforegoing description and examples are for purposes of illustration andnot limitation of the following claims.

What is claimed is:
 1. A method for the production of mycolactones,which method comprises cultivation of Mycobacterium ulcerans in adispersed suspension culture by fermentation or in spinner flasks. 2.The method of claim 1, wherein the mycolactones are produced inconcentrations greater than that observed in T flasks.
 3. The method ofclaim 1, wherein said cells are cultivated in M7H9 medium.
 4. The methodof claim 3, wherein said medium is enriched.
 5. The method of claim 4,wherein said medium is enriched by addition of egg yolk.
 6. A method forthe purification of mycolactones that comprises extraction of media inwhich Mycobacterium ulcerans has been cultivated and chromatographicseparation of said extract.
 7. Mycolactone C in substantially purifiedform.
 8. Mycolactone D in substantially purified form.